Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal [AutoCUT&Tag]

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor are appealing towards inducing HIV reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 in both cell line models and in CD4 T cells from virally suppressed PLWH improves reactivation at the transcriptional level and is more specific than AZD5582 & I-BET151 treatment alone. INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observe more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151 indicating the combination may specifically overcome an elongation block to reactivation. We also observe viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 controls full-length HIV RNA and virus production ex vivo.